1. Field of the Invention
The field of the invention relates generally to methods for expressing LIM mineralization proteins in a host cell. More specifically, the field of the invention relates to expression and purification of intracellular mediators involved in the osteogenic factor signaling cascade during osteoblast differentiation. The field of invention also relates to methods of improving the nuclear localization of osteogenic factors in suitable cells. Finally, the field of invention relates to transfecting host cells such as intervertebral disc cells with a nucleic acid encoding a LIM mineralization protein.
2. Background of the Technology
Osteoblasts are thought to differentiate from pluripotent mesenchymal stem cells. The maturation of an osteoblast results in the secretion of an extracellular matrix which can mineralize and form bone. The regulation of this complex process is not well understood but is thought to involve a group of signaling glycoproteins known as bone morphogenetic proteins (BMPs). These proteins have been shown to be involved with embryonic dorsal-ventral patterning, limb bud development, and fracture repair in adult animals. B. L. Hogan, Genes & Develop., 10, 1580 (1996).
BMPs are members of the Transforming growth factor-beta superfamily (TGF-β) proteins that play a role in maturation of osteoblasts and the bone regulation pathways. TGF-β secreted proteins has a spectrum of activities in a variety of cell types at different stages of differentiation. However, differences in physiological activity between these closely related molecules have not been clarified. D. M. Kingsley, Trends Genet., 10, 16 (1994).
In addition to extracellular signals, such as the BMPs, intracellular signals or regulatory molecules also play a role in the cascade of events leading to formation of new bone. Examples of such regulatory osteogenic molecules include LIM mineralization proteins (LMPs), Runt-Related Transcription Factor (Runx-2), Drosophila distalles (Dlx), and Osterix (Osx).
It is common knowledge that the differentiation process of osteoblasts requires a complex coordination of all such osteogenic factors including BMPs, other members of the transforming TGF-β superfamily, LMPs, Dlx, Runx-2, Osx are key osteogenic factors that play critical roles in the BMP pathway.
To better discern the unique physiological role of different factors studies are required to better understand the nature of their interaction. We have performed many studies on the nature of BMP signaling protein pathways. We have recently compared the potency of BMP-6 with that of BMP-2 and BMP-4, for inducing rat calvarial osteoblast differentiation. Boden, et al., Endocrinology, 137, 3401 (1996). We studied this process in first passage (secondary) cultures of fetal rat calvaria that require BMP or glucocorticoid for initiation of differentiation. In this model of membranous bone formation, glucocorticoid (GC) or a BMP will initiate differentiation to mineralized bone nodules capable of secreting osteocalcin, the osteoblast-specific protein. This secondary culture system is distinct from primary rat osteoblast cultures which undergo spontaneous differentiation. In this secondary system, glucocorticoid resulted in a ten-fold induction of BMP-6 mRNA and protein expression which was responsible for the enhancement of osteoblast differentiation. Boden, et al., Endocrinology, 138, 2920 (1997).
Other investigations have also been performed to assess the relationship between other osteogenic factors and BMPs. Bourque et al elaborated on Runx-2 critical role in the differentiation of cells toward an osteoblastic pathway. Bourque et al, Expression of four growth factors during fracture repair. Int. J. Dev. Biol, 1993:37:573-9. Runx2 is a transcription factor that belongs to the Runx family. Komori, T., et al. 1997. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 89:755-764; Fujita et al (2004) “Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling.” J. Cell Biol. 166:85-95.
Runx2-deficient (Runx2−1−) mice completely lack bone formation owing to the absence of osteoblasts. Runx2 determines the osteoblast lineage from multipotent mesenchymal cells, induces osteoblastic differentiation at the early stage, and inhibits it at the late stage. Further, Runx2 has been shown to induce alkaline phosphatase (ALP) activity, expression of bone matrix protein genes, and mineralization in immature mesenchymal cells and osteoblastic cells in vitro. Chondrocyte differentiation is also disturbed in Runx2−/− mice.
Overexpression of Runx2 or the dominant-negative (dn) form of Runx2 (dn-Runx2) in chondrocytes accelerates or decelerates chondrocyte maturation, respectively, indicating that Runx2 is a positive regulatory factor in chondrocyte maturation. Further, introduction of dn-Runx2 inhibited cell condensation in insulin-induced chondrogenesis of ATDC5 cells. Thus, Runx2 plays crucial roles in osteoblast and chondrocyte differentiation.
Dlx proteins have been implicated to play major role in osteogenesis as well. Ryoo, H. M., et al. (1997) “Stage-specific expression of Dlx-5 during osteoblast differentiation: involvement in regulation of osteocalcin gene expression.” Mol. Endocrinol. 11, 1681-1694. It has been shown that the mammalian homologs of Dlx 5 and 6 are homeobox genes essential for craniofacial and skeletal development. Dlx5 is a target gene for BMPs that regulate osteogenesis and dorsoventral patterning and targeted gene inactivation of Dlx 5 and 6 results in severe skeletal abnormalities leading to prenatal lethality. Sandhu et al. “Evaluation of rhBMP-2 with an OPLA carrier in a canine posterolateral (transverse process) spinal fusion model.” Spine 1995; 20:2669-82.
Osx is a novel zinc finger containing transcription factor expressed by osteoblasts and required for endochondral and intra membranous bone formation. Nakashima, K., et al, (2002) “The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation.” Cell 108:17-29. Osterix-null mice have normal cartilage development but fail to develop mineralized skeleton. Yasko et al. the Healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic, histological, and biomechanical study in rats. J Bone Joint Surg Am, 1992; 74:659-70.
Another broad class of intracellular regulatory molecules are the LIM proteins, which are so named because they possess a characteristic structural motif known as the LIM domain. Viggeswarapu M, Boden S D et al. (2001) “Adenoviral delivery of LIM mineralization protein-1 induces new-bone formation in vitro and in vivo.” J Bone & Joint Surg Am. 83:364-376. The LIM domain is a cysteine-rich structural motif composed of two special zinc fingers that are joined by a 2-amino acid spacer. Some proteins have only LIM domains, while others contain a variety of additional functional domains. LIM proteins form a diverse group, which includes transcription factors and cytoskeletal proteins. The primary role of LIM domains appears to be in mediating protein-protein interactions, through the formation of dimers with identical or different LIM domains, or by binding distinct proteins.
In LIM homeodomain proteins, that is, proteins having both LIM domains and a homeodomain sequence, the LIM domains function as negative regulatory elements. LIM homeodomain proteins are involved in the control of cell lineage determination and the regulation of differentiation, although LIM-only proteins may have similar roles. LIM-only proteins are also implicated in the control of cell proliferation since several genes encoding such proteins are associated with oncogenic chromosome translocations.
Humans and other mammalian species are prone to diseases or injuries that require the processes of bone repair and/or regeneration. For example, treatment of fractures would be improved by new treatment regimens that could stimulate the natural bone repair mechanisms, thereby reducing the time required for the fractured bone to heal. In another example, individuals afflicted with systemic bone disorders, such as osteoporosis, would benefit from treatment regimens that would result in systemic formation of new bone. Such treatment regimens would reduce the incidence of fractures arising from the loss of bone mass that is a characteristic of this disease.
For at least these reasons, extracellular factors, such as the BMPs, have been investigated for the purpose of using them to stimulate formation of new bone in vivo. Despite the early successes achieved with BMPs and other extracellular signaling molecules, their use entails a number of disadvantages.
For example, relatively large doses of purified BMPs are required to enhance the production of new bone, thereby increasing the expense of such treatment methods. Furthermore, extracellular proteins are susceptible to degradation following their introduction into a host animal. In addition, because they are typically immunogenic, the possibility of stimulating an immune response to the administered proteins is ever present.
Due to such concerns, it would be desirable to have available treatment regimens that use an intracellular signaling molecule that can induce new bone formation. Advances in the field of gene therapy now make it possible to introduce into osteogenic precursor cells, that is, cells involved in bone formation, or peripheral blood leukocytes, nucleotide fragments encoding intracellular signals that form part of the bone formation process. Gene therapy for bone formation offers a number of potential advantages: (1) lower production costs; (2) greater efficacy, compared to extracellular treatment regimens, due to the ability to achieve prolonged expression of the intracellular signal; (3) it would by-pass the possibility that treatment with extracellular signals might be hampered due to the presence of limiting numbers of receptors for those signals; (4) it permits the delivery of transfected potential osteoprogenitor cells directly to the site where localized bone formation is required; and (5) it would permit systemic bone formation, thereby providing a treatment regimen for osteoporosis and other metabolic bone diseases.
In addition to diseases of the bone, humans and other mammalian species are also subject to intervertebral disc degeneration, which is associated with, among other things, low back pain, disc herniation, and spinal stenosis. Disc degeneration is associated with a progressive loss of proteoglycan matrix. This may cause the disc to be more susceptible to bio-mechanical injury and degeneration. Accordingly, it would be desirable to have a method of stimulating proteoglycan and/or collagen synthesis by the appropriate cells, such as, for example, cells of the nucleus pulpous, cells of the annulus fibrosis, and cells of the intervertebral disc.